LOCUS       pTwist_Lenti_TRE        7673 bp DNA     circular SYN 22-OCT-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7673)
  AUTHORS   siwong
  TITLE     Direct Submission
  JOURNAL   Exported Oct 28, 2024 from SnapGene 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..315
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    80..98
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    116..134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    152..170
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    187..205
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    223..241
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    336^337
                     /label=Your gene here for 6xHis_tag
     CDS             337..354
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    337..354
                     /label=Removed for No_tag
     promoter        364..874
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             884..1480
                     /codon_start=1
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             1481..1534
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1535..2152,2153..2158,2159..2194,2195..2197,2198..2233,
                     2234..2236,2237..2272,2273..2281)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    2308..2896
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(2779..2790)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2995..3228
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3300..3421
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      3461..3596
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3596..3667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3668..3736,3737..4528)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4699..5287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5575..5596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5611..5616,5617..5634,5635..5641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5649..5665
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5673..5689
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5710..5728
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5756..5982
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5983..6163
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6210..6335
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6828..7061
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7246..7290
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7503..7620
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
       61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
      121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
      181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
      241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
      301 aaccgtcaga tcgcctggag aattggctag catcagcacc accaccacca ccactaagga
      361 tccggggttg gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg
      421 ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac
      481 gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct
      541 tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca
      601 aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc
      661 agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag
      721 cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt
      781 cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc
      841 cctcgttgac cgaatcaccg acctctctcc ccagcaattc accatgaccg agtacaagcc
      901 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
      961 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     1021 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     1081 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     1141 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     1201 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     1261 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     1321 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     1381 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     1441 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc gaaggtagag gttctctcct
     1501 cacttgtggt gatgttgaag aaaaccctgg tccaatgtct agactggaca agagcaaagt
     1561 cataaacgga gctctggaat tactcaatgg tgtcggtatc gaaggcctga cgacaaggaa
     1621 actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg
     1681 ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc acttctgccc
     1741 cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat accgctgtgc
     1801 tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca
     1861 gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa
     1921 cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggaaca
     1981 ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact
     2041 tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct tccttttcgg
     2101 cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccgac
     2161 cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg acgactttga
     2221 ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc tccccgggta
     2281 actaagtaag gatcgatcca agatatcaat caacctctgg attacaaaat ttgtgaaaga
     2341 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     2401 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     2461 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     2521 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     2581 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     2641 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     2701 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     2761 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     2821 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     2881 tgggccgcct ccccgcctgt ttcgcctcgg cgtccggact agaggtacct ttaagaccaa
     2941 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     3001 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     3061 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     3121 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     3181 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     3241 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga
     3301 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3361 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3421 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc
     3481 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3541 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctttc
     3601 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3661 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3721 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3781 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3841 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3901 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3961 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4021 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4081 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4141 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4201 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4261 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4321 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4381 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4441 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4501 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4561 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4621 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4681 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4741 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4801 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4861 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4921 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4981 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5041 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5101 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5161 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5221 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5281 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5341 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5401 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5461 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5521 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     5581 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     5641 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     5701 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag
     5761 tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta
     5821 caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc
     5881 ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat
     5941 tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac
     6001 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     6061 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     6121 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
     6181 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
     6241 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
     6301 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
     6361 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
     6421 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
     6481 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     6541 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     6601 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     6661 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     6721 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     6781 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     6841 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     6901 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     6961 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     7021 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     7081 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     7141 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     7201 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     7261 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     7321 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     7381 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     7441 cacctcccaa ccccgagggg acaattctcg acctcgagac aaatggcagt attcatccac
     7501 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     7561 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     7621 cgggtttatt acagggacag cagagatcca ctttggccgc gaatcgatat gtc
//
